With the use of experimentally reconstructed steady-state cell-free systems, we propose to evaluate in a series of model systems (a) the nature of the apparent effects of Ca2+ and its segregation on e.g. fluxes through central branch-points in mitochondrial disposition of pyruvate (carboxylation and decarboxylation, vectorial movement of citrate); (b) the signals mediating persisting changes in mitochondria which result from pretreatment with glucagon or Alpha-adrenergic agonists; the extent to which these responses are correlated with altered CA2+ movements; (c) the degree to which changes in membrane-associated and bioenergetic parameters (compartmented ATP/ADP and redox potentials) and correlated under steady-state metabolizing conditions with effects of Ca2+ and by in situ endocrine treatments on mitochondria subsequently processed; (d) the quantitative role of citrate in control of glycolytic flux in muscle when other putative effectors are held constant in an 'open' system; and the influence of dietary-endocrine state of animals on citrate movements from liver mitochondria; and (e) possibilities for detecting and identifying extrinsically imposed signals generated by glucagon and Alpha-adrenergic agonists in there cell-free steady-state systems containing endocrine-responsive plasma membranes on mitochondrial function. These studies potentially have far-reaching implications in our understanding of normal- and abnormal manifestations of intrinsic (intracellular) and extrinsic (imposed from without) controls. The model systems are experimentally unique since vectorially poised multicomponent and multicompartment processes are integrated into steady-state 'open' systems resembling the intact cell. With a rat muscle preparation perfused with a synthetic medium we propose to extend studies of the quantitative flux of carbon and nitrogen under conditions in which these compounds serve as glucogenic precursors. Endocrine effects on this balance will also be evaluated. This, and the cell-free systems described, provide a framework, at several levels of organization, to evaluate intracellular, intraorgan, and interorgan metabolic fluxes under simulated physiological and pathological conditions, and to evaluate intrinsic and extrinsic influences on these processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK013939-16
Application #
3225171
Study Section
Biochemistry Study Section (BIO)
Project Start
1977-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1988-03-31
Support Year
16
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Schild, L; Blair, P V; Davis, W I et al. (1999) Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels. Biochim Biophys Acta 1413:14-20
Dietzen, D J; Davis, E J (1994) Excess membrane cholesterol is not responsible for metabolic and bioenergetic changes in AS-30D hepatoma mitochondria. Arch Biochem Biophys 309:341-7
Dietzen, D J; Davis, E J (1993) Oxidation of pyruvate, malate, citrate, and cytosolic reducing equivalents by AS-30D hepatoma mitochondria. Arch Biochem Biophys 305:91-102
Davis, E J; Davis-van Thienen, W I (1991) An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation. Arch Biochem Biophys 289:184-6
Kunz, W S; Davis, E J (1991) Control of reversible intracellular transfer of reducing potential. Arch Biochem Biophys 284:40-6
Davis, E J; Davis-van Thienen, W I (1989) Force-flow and back-pressure relationships in mitochondrial energy transduction: an examination of extended state 3-state 4 transitions. Arch Biochem Biophys 275:449-58
Scislowski, P W; Bremer, J; van Thienen, W I et al. (1989) Heart mitochondria metabolize 3-methylthiopropionate to CO2 and methanethiol. Arch Biochem Biophys 273:602-5